色氨酸肠道菌群代谢物通过激活“外源性化合物感受器”参与骨质疏松症发生的机制研究

Osteoporosis is a common clinical disorder, which is resulted from imbalance between bone formation by osteoblasts and bone resorption by osteoclasts. Gut microbiota is the complex community of microorganisms that live in the digestive tracts of humans that are involved in many pathology and pathogenesis processes. Tryptophan can be metabolized by the gut bacteria into indole derivatives, such as indole (Ind), indoxyl-3-sulfate (I3S), indole-3-propionic acid (IPA), indole-3-aldehyde (I3A), indole-3-acetic acid (I3AA). In our preliminary experiments we found that serum I3AA levels from postmenopausal women were lower than that from premenopausal women and Pearson correlation analysis indicated that BMD (bone mineral density) was significantly correlated with serum I3AA level. Further we found that part of these indole derivatives modulated osteoclasts differentiation and function. RT-qPCR results showed microbiota derived tryptophan derivatives activated xenobiotic sensors including PXR, AhR and CAR. We thus speculate that menopause caused shift of composition of gut microbiota and subsequently altered gut microbiota derived tryptophan metabolites are involved in pathogenesis of osteoporosis. In this project the research team will try to investigate changes of composition of gut microbiota and related tryptophan metabolites. In this way we want to elucidate the effect of tryptophan metabolism related gut microbiota and tryptophan metabolites on bone metabolism.

骨质疏松症是一种临床常见疾病，主要病理机制为成骨细胞参与的骨形成和破骨细胞参与的骨吸收失衡。肠道菌群是一类人体的共生生物，参与多种生理病理过程。色氨酸经肠道菌群代谢产生多种吲哚衍生物。申请人前期实验发现，绝经后血清吲哚乙酸（I3AA）水平明显低于绝经前，且I3AA水平和女性骨密度具有显著相关性。申请人进一步实验证实，部分吲哚衍生物调节破骨细胞分化和功能，且吲哚、吲哚丙酸、吲哚甲酸、吲哚乙醛可明显激活“外源性化合物感受器”（包括PXR/AhR/CAR）。基于上述结果，申请人推测，绝经前后色氨酸代谢相关肠道菌群丰度改变、血清色氨酸代谢物水平异常参与骨质疏松的发生。在本课题中，申请人团队拟通过进一步实验在绝经前后女性和卵巢切除/假手术小鼠模型中明确色氨酸代谢相关肠道菌群及血清代谢物变化、色氨酸代谢肠道菌群/代谢物对小鼠骨量的调控作用及其分子机制。

破骨细胞介导的骨吸收功能参与骨代谢动态平衡过程，破骨细胞数量过多或功能过度活跃导致骨质疏松症等多种骨疾病发生。肠道菌群及其代谢物在多种疾病中发挥关键作用，也与骨代谢平衡密切相关。色氨酸代谢物是肠道菌群主要代谢物之一，其是否影响破骨细胞的分化及机制尚未有相关报道。本课题旨在研究肠道菌群和色氨酸代谢物对破骨细胞的作用和调控分子机制，为治疗骨质疏松症等破骨细胞相关病提供潜在的干预靶点和新的治疗策略。. 本项目主要通过16sRNA微生物测序检测正常小鼠和OVX小鼠肠道菌群变化，同时检测血清中色氨酸代谢物差异；使用色氨酸代谢物IPA干预BMMs以验证对破骨细胞形成和功能、破骨分化过程中的关键转录因子以及破骨细胞标记基因的表达、NF-κB以及MAPK通路的影响。通过OVX小鼠行IPA/色氨酸/生孢梭菌联合色氨酸灌胃探究色氨酸代谢相关肠道菌群及代谢物对OVX引起的骨量丢失的影响。. 16sRNA结果表明OVX小鼠梭菌属丰度明显减少，同时血清中其色氨酸代谢物IPA含量减少。体外实验证实IPA对BMMs无毒副作用，其可以明显抑制破骨细胞分化及其骨吸收能力，降低破骨分化过程中重要基因的表达水平。通过灌胃给予IPA可以减缓OVX小鼠的骨量下降，骨组织内破骨细胞被明显抑制，表明IPA在动物体内也可以抑制破骨细胞形成。同时IPA可以明显抑制NF-κB和MAPK通路中关键蛋白磷酸化水平。qPCR结果表明IPA能够上调PXR下游基因的表达(CYP3A11, MDR1, UGT1A1), 通过免疫共沉淀和免疫荧光等实验，发现 IPA 通过上调 PXR 的表达，进而促进 PXR 结合游离的p65抑制其入核发挥生物学效应，从而抑制 NF-κB 信号通路的激活。. 通过本项目的实施，我们发现了生孢梭菌和色氨酸代谢物IPA对破骨细胞分化和功能的抑制性作用，在体阻碍小鼠骨量丢失，并发现了可能的分子机制，从而为骨质疏松症等因破骨细胞过度活跃导致的骨疾病的防治提供潜在的干预靶点和治疗策略。